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Lookup NU author(s): Dr Don Mahad, Dr Iryna Ziabreva, Graham Campbell, Dr Julie Murphy, Dr Amy Reeve, Professor Laura GreavesORCiD, Emeritus Professor Doug Turnbull
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Cytochrome c oxidase or mitochondrial respiratory chain complex IV is where over 90% of oxygen is consumed. The relationship between complex IV activity and mitochondrial proteins, which provides a guide to understanding the mechanisms in primary mitochondrial disorders, has been determined by histochemistry (activity) and immunohistochemistry in serial sections. In the central nervous system (CNS), mitochondrial activity and immunoreactivity have been determined in populations of cells in serial sections as capturing cells in more than one section is difficult. In this report we describe a method to determine complex IV activity in relation to mitochondrial proteins at a single cell level in the CNS. We performed complex IV histochemistry and immunohistochemistry consecutively in snap frozen sections. Although the product of complex IV histochemistry reduces the sensitivity of standard immunohistochemistry (secondary antibody and ABC method) the biotin-free Menapath polymer detection system (A. Menarini Diagnostics, Wokingham, UK) enables mitochondrial proteins to be detected following complex IV histochemistry. The co-occurring chromogens may then be separately visualised and analysed using multi-spectral imaging (Nuance system CRI, Woburn, MA). Our technique is applicable for exploring mitochondrial defects within single cells in a variety of CNS disorders and animal models of those diseases. (C) 2009 Elsevier B.V. All rights reserved.
Author(s): Mahad DJ, Ziabreva I, Campbell G, Laulund F, Murphy JL, Reeve AK, Greaves L, Smith KJ, Turnbull DM
Publication type: Article
Publication status: Published
Journal: Journal of Neuroscience Methods
Year: 2009
Volume: 184
Issue: 2
Pages: 310-319
ISSN (print): 0165-0270
ISSN (electronic): 1872-678X
Publisher: Elsevier BV
URL: http://dx.doi.org/10.1016/j.jneumeth.2009.08.020
DOI: 10.1016/j.jneumeth.2009.08.020
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